The invention relates to the fields of immunology and Coeliac disease. The invention in particular relates to the antibodies generated against different gluten proteins involved in Coeliac disease and uses thereof for the detection of those proteins in different backgrounds as there are/ for instance; protein (digests) of wheat, food products, wheat starch hydrolysates, protein hydrolysates of wheat proteins, raw materials and semi-finished materials used in food industry.
Coeliac disease (CD) is a permanent intolerance for wheat gluten proteins, a complex mixture of storage proteins1. Similar (gluten-like) proteins are present in other cereals like barley, rye, oats and triticale (a hybrid of wheat and rye). Typical symptoms observed in CD patients are chronic diarrhea, malnutrition, anemia, fatigue and growth retardation. These symptoms are the result of a lesion in the small intestine characterized by (sub) total villous atrophy and increased numbers of intraepithelial lymphocytes2.
It is now generally accepted that CD is an immune disease caused by T cells recognizing gluten derived peptides presented by HLA-DQ2 or HLA-DQ8 molecules. Such gluten-specific, CD4+, HLA-DQ2 or HLA-DQ8 restricted, T lymphocytes can be isolated from small intestinal biopsies of patients but not of controls34567. T cell stimulatory peptides have been identified by us an others, and these originate from proline and glutamine rich regions in α-gliadin, γ-gliadin, and low (LMW) and high molecular weight (HMD) glutenins8910111213. Modification of these peptides, due to the activity of the enzyme tTG is in the majority of cases required for, or enhances the gluten specific T cell response. tTG activity converts glutamine residues in gluten peptides into glutamic acid which facilitates gluten peptide binding to HLA-DQ2 or HLA-DQ814151617. This provides an explanation for the observation that the presence of these molecules predisposes to disease development31819672021.
Omission of gluten from the diet of CD patients leads to disappearance of CD symptoms and full recovery of the small intestine. The Codex Alimentarius defines gluten free foods as those whose gluten contents are below 200 ppm (200 mg gluten/100 g of food), which is equivalent to 100 ppm of gliadins To further ensure safety for CD patients, it has been proposed to decrease this level to 20 ppm22.
The accurate detection of gluten however, is complicated since gluten is composed of two different protein families, the LMW and HMW molecular weight glutenins and gliadins. The latter can be further subdivided into α-, γ-, and ω-gliadins. Moreover, each subgroup of the gliadins consists of a mixture of highly similar but distinct proteins (for a recent review23). Another complication for the detection of gluten proteins is that they can be present in the food products both as intact protein and as small protein fragments. In order to be used in gluten free food products, wheat starch with remaining low protein content is hydrolyzed chemically or enzymatically. During this process the gluten proteins are digested into small peptides and amino acids. When this hydrolysis is incomplete, protein fragments will remain with sizes that are big enough to stimulate T cells. Moreover, protein hydrolysates are widely used in the food industry including hydrolysates originating from wheat that may also contain gluten and smaller fragments thereof.
For the detection of gluten two commercially available methods are currently available, both based on a sandwich Elisa system. In one assay ω-gliadins24 are detected, while in the other assay both α-, γ- and ω-gliadins are detected25. However, when used for the screening of the safety of food used by CD patients, the methods have three major disadvantages. First, the assays are not specific for detection of T cell stimulatory sequences in gluten. Secondly, the methods that are based on a sandwich Elisa are not suitable for the detection of small peptides i.e. of sizes recognizable by T cells. Thirdly, the methods do not detect Low Molecular Weight and High Molecular Weight glutenin proteins. Consequently there is an urgent need for better assays that detect those sequences in gluten, both originating from gliadin and glutenin, that stimulate gluten specific T cells in the intestine of CD patients on both intact proteins and on peptides of sizes that can be recognized by T cells.